Two-color synergistic interaction colored microspheres as well as preparation method and application thereof

Abstract

The invention relates to the technical field of immunochromatography, in particular to a double-color synergistic interaction colored microsphere and a preparation method and application thereof, and the specific method comprises the following steps: preparing a red silicon dioxide core by a Stber method, and then coating the red silicon dioxide core with a blue polystyrene shell layer. The double-color synergistic interaction colored microspheres prepared by the invention can be used for high-sensitivity single-linkage detection or multi-linkage detection of infection markers. According to the colored microspheres prepared by the method, due to the synergistic effect of blue and red colors, the sensitivity is improved by one order of magnitude compared with that of colored microspheres embedded with single dye, and the detection accuracy and sensitivity of high-sensitivity detection items are improved.

Description

Technical Field

[0001] The present invention relates to the field of immunochromatography technology, and specifically relates to a dual-color synergistically enhanced colored microsphere and a preparation method and application thereof. Background Art

[0002] Immunochromatography is a simple, rapid, and highly specific diagnostic method widely used in clinical diagnosis, food safety, drug testing, and environmental pollution. It primarily relies on immunochromatographic test strips as a carrier, using solid-phase markers to qualitatively, semi-qualitatively, or quantitatively detect the substance in the sample. While traditional colloidal gold immunochromatographic test strips offer advantages such as ease of use and low cost, they suffer from low sensitivity and poor accuracy, making them difficult to meet the needs of quantitative testing.

[0003] To improve detection sensitivity, researchers have developed a variety of novel signal probes, such as fluorescent latex microspheres, quantum dot fluorescent microspheres, and time-resolved microspheres. These fluorescent probes overcome the limitations of traditional colloidal gold test strips by enhancing fluorescence signals and improving optical stability. However, these fluorescent probes still have some drawbacks, such as requiring an additional UV excitation light source and only being able to produce a single signal output.

[0004] In recent years, colored microsphere technology has become the core engine for immunochromatography to move from qualitative to quantitative through innovations such as internal embedding dyeing process, precise particle size control and high-density surface modification. This technology combines dyes with polymer microspheres to form a composite material with high sensitivity and specificity, and is widely used in fields such as biological agglutination tests and lateral chromatography. However, the existing methods for preparing colored microspheres still have some problems, such as low dye loading rate, difficulty in removing surfactants, and difficulty in controlling the core-shell structure. In particular, for some highly sensitive detection items, such as the preparation of blue and purple microspheres, existing technologies are difficult to meet the needs, affecting the accuracy and sensitivity of the test results. Therefore, the development of a method for preparing colored microspheres with high sensitivity, uniform particle size, high dye loading rate and non-leakage is of great significance for promoting the application and development of immunochromatography technology.

[0005] Regarding solving the sensitivity and color depth problems of colored microspheres in the field of immunochromatographic detection, there are currently some patent documents. For example, CN116550311A discloses a method and application of synthesizing colored microspheres by a hydrothermal synthesis method, which prepares monodisperse polystyrene latex microspheres by emulsion polymerization, and mixes an oil-soluble dye with a blank polystyrene latex microsphere, and adopts a hydrothermal synthesis method to synthesize colored latex microspheres in a high temperature and high pressure step. The method is simple to operate, does not use an emulsifier, and the prepared colored microspheres have uniform particle size, good dispersibility, high dye loading rate and are not easy to leak. However, the colored microspheres prepared by this method have the problem that the single color microspheres are not deeply colored, and in particular, the microspheres of a single color such as red, blue, and purple microspheres are difficult to meet the needs of high-sensitivity detection projects.

[0006] Human myxovirus resistance protein A (MxA) is a cytoplasmic protein expressed by interferon type I / III in human cells. It has GTPase activity and a molecular weight of approximately 75 kDa. Even small amounts of virus can induce MxA expression, resulting in broad-spectrum antiviral activity and a highly sensitive and specific marker for viral infection. MxA protein measurement is crucial for preventing the overuse of antibiotics and improving the diagnosis of clinical infectious diseases. Combined detection of MxA with other inflammatory markers, such as C-reactive protein (CRP), procalcitonin (PCT), pancreatic stone protein (PSP), interleukin-6 (IL-6), and heparin-binding protein (HBP), can significantly improve the clinical sensitivity and specificity for distinguishing viral from bacterial infections.

[0007] In summary, existing methods for preparing colored microspheres often produce microspheres of a single color (such as red, blue, or purple) with limited depth, making them difficult to meet the requirements of high-sensitivity testing, thus impacting the accuracy and sensitivity of test results. Acute respiratory infections are common in respiratory clinics, and early differentiation between bacterial and viral infections is crucial for optimal treatment. Combining the detection of MxA protein with CRP, PCT, PSP, IL-6, and HBP can improve diagnostic accuracy, prevent antibiotic overuse, optimize treatment strategies, reduce the risk of drug resistance, and ensure patient safety. Summary of the Invention

[0008] The purpose of the present invention is to provide a dual-color synergistic colored microsphere and a preparation method and application thereof.

[0009] To achieve the above object, the present invention provides the following technical solutions: A method for preparing dual-color synergistic colored microspheres comprises the following steps: (1) Preparation of silica red microsphere core: add pure water and anhydrous ethanol in a reaction container, add red oil-soluble dye, after ultrasonic dissolution, fix the reaction container on an electric stirrer for stirring, add ammonia water, after a certain time, add silane coupling agent TEOS, react at room temperature overnight, after the reaction is completed, centrifuge to remove the mother liquor, add pure water and ultrasonic dispersion for standby; (2) Coating of blue polymer shell: add pure water in a reaction container, remove oxygen by nitrogen blowing, add anionic surfactant and stir to dissolve, add the silica red microsphere core prepared in step (1); weigh the blue oil-soluble dye and dissolve in the purified mixed solution of styrene and functional monomer, slowly add into the reaction container, emulsify at high speed, heat to 60℃, add potassium persulfate, react at 75-85℃ for 4-12h.

[0010] In step (1), the volume ratio of pure water to anhydrous ethanol is 1:4-9, and the red oil-soluble dye includes solvent red or dispersion red, and the mass ratio of red oil-soluble dye to silane coupling agent TEOS is 1:2-3.

[0011] In step (2), the blue oil-soluble dye includes dispersion blue and solvent blue, and the mass ratio of blue oil-soluble dye to all monomers is 1:10-50; the anionic surfactant is sodium dodecyl sulfate; the functional monomer is methacrylic acid or acrylic acid, preferably, the functional monomer is methacrylic acid. The volume ratio of styrene to functional monomer is 1:0.04-0.2.

[0012] The bicolored synergistic color microspheres prepared by the above preparation method can be used for high-sensitivity single-link detection or multi-link detection of infection markers.

[0013] The infection markers are viral infection markers and bacterial infection markers, the viral infection markers are MxA proteins, and the bacterial infection markers include CRP, PCT, PSP, IL-6 and HBP.

[0014] Compared with the prior art, the bicolored synergistic color microspheres prepared by the above preparation method have the following beneficial effects: The color microspheres prepared by the above preparation method have a bicolored synergistic effect of blue and red, and the sensitivity is one order of magnitude higher than that of the color microspheres embedded with a single dye, thereby improving the detection accuracy and sensitivity of high-sensitivity detection projects. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The photos of the color microspheres prepared in Examples 1-3 and Comparative Examples 1-2. DETAILED DESCRIPTION

[0016] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0017] Example 1 Preparation of dual-color synergistic colored microspheres by emulsion polymerization (1) Preparation of red silica microsphere cores: Add 10 mL of pure water and 90 mL of anhydrous ethanol to a three-necked flask, add 2 g of Solvent Red 26, and ultrasonically dissolve it. Then, fix the three-necked flask on an electric stirrer and stir it. Add 5 mL of ammonia water. After 10 minutes, add 5 mL of silane coupling agent TEOS and react at room temperature overnight. After the reaction is completed, centrifuge to remove the mother liquor, add pure water and ultrasonically disperse it for later use.

[0018] (2) Coating with a blue polymer shell: Add 200 mL of pure water to a three-necked flask, deoxygenate with nitrogen, add 50 mg of SDS and stir to dissolve, then add the red silica microsphere core prepared in step (1). Weigh 0.3 g of Sudan Blue B and dissolve it in a mixture of 10 mL of purified styrene and 2 mL of methacrylic acid. Slowly add it to the reaction flask and stir at high speed to emulsify. Heat to 60°C, add 120 mg of potassium persulfate, and react at 80°C for 8 h.

[0019] Products such as Figure 1 shown.

[0020] Example 2 Preparation of dual-color synergistic colored microspheres by emulsion polymerization (1) Preparation of red silica microsphere cores: Add 10 mL of pure water and 90 mL of anhydrous ethanol to a three-necked flask, add 2 g of Solvent Red 26, and ultrasonically dissolve it. Then, fix the three-necked flask on an electric stirrer and stir it. Add 5 mL of ammonia water. After 10 minutes, add 5 mL of silane coupling agent TEOS and react at room temperature overnight. After the reaction is completed, centrifuge to remove the mother liquor, add pure water and ultrasonically disperse it for later use.

[0021] (2) Coating with a blue polymer shell: Add 200 mL of pure water to a three-necked flask, deoxygenate with nitrogen, add 50 mg of SDS and stir to dissolve, then add the red silica microsphere core prepared in step (1). Weigh 0.5 g of Sudan Blue B and dissolve it in a mixture of 10 mL of purified styrene and 2 mL of methacrylic acid. Slowly add it to the reaction flask and stir at high speed to emulsify. Heat to 60°C, add 120 mg of potassium persulfate, and react at 80°C for 8 h.

[0022] Products such as Figure 1 shown.

[0023] Example 3 Preparation of dual-color synergistic colored microspheres by emulsion polymerization (1) Preparation of red silica microsphere cores: Add 10 mL of pure water and 90 mL of anhydrous ethanol to a three-necked flask, add 2 g of Solvent Red 26, and ultrasonically dissolve it. Then, fix the three-necked flask on an electric stirrer and stir it. Add 5 mL of ammonia water. After 10 minutes, add 5 mL of silane coupling agent TEOS and react at room temperature overnight. After the reaction is completed, centrifuge to remove the mother liquor, add pure water and ultrasonically disperse it for later use.

[0024] (2) Coating with a blue polymer shell: Add 200 mL of pure water to a three-necked flask, deoxygenate with nitrogen, add 50 mg of SDS and stir to dissolve, then add the red silica microsphere core prepared in step (1). Weigh 1 g of Sudan Blue B and dissolve it in a mixture of 10 mL of purified styrene and 2 mL of methacrylic acid. Slowly add it to the reaction flask and stir at high speed to emulsify. Heat to 60°C, add 120 mg of potassium persulfate, and react at 80°C for 8 h.

[0025] Products such as Figure 1 shown.

[0026] In order to highlight the beneficial effects of the present invention, the following comparative example experiments are given.

[0027] Comparative Example 1 Preparation of red microspheres (1) Add 10 mL of pure water and 90 mL of anhydrous ethanol to a three-necked flask, add 2 g of Solvent Red 26, and ultrasonically dissolve it. Then, fix the three-necked flask on an electric stirrer and stir it. Add 5 mL of ammonia water. After 10 minutes, add 5 mL of silane coupling agent TEOS and react at room temperature overnight. After the reaction is completed, centrifuge to remove the mother liquor, add pure water and ultrasonically disperse it for later use.

[0028] (2) Add 200 mL of pure water to a three-necked flask, pass nitrogen to deoxygenate, add 50 mg of SDS and stir to dissolve, add the red silica microsphere core prepared in step (1); dissolve 0.5 g of Solvent Red 26 in 10 mL of purified styrene and 2 mL of methacrylic acid mixed solution, slowly add to the reaction flask, and stir at high speed to emulsify. Heat to 60°C, add 120 mg of potassium persulfate, and react at 80°C for 8 hours. The product is as follows: Figure 1 shown.

[0029] Comparative Example 2 Preparation of blue microspheres (1) Add 10 mL of pure water and 90 mL of anhydrous ethanol to a three-necked flask, add 2 g of Sudan Blue B, and ultrasonically dissolve it. Then, fix the three-necked flask on an electric stirrer and stir it. Add 5 mL of ammonia water. After 10 minutes, add 5 mL of silane coupling agent TEOS and react at room temperature overnight. After the reaction is completed, centrifuge to remove the mother liquor, add pure water and ultrasonically disperse it for later use.

[0030] (2) Add 200 mL of pure water to a three-necked flask, pass nitrogen to deoxygenate, add 50 mg of SDS and stir to dissolve, then add the silica microsphere core prepared in step (1); dissolve 0.5 g of Sudan Blue B in 10 mL of purified styrene and 2 mL of methacrylic acid mixed solution, slowly add to the reaction flask, and stir at high speed to emulsify. Heat to 60°C, add 120 mg of potassium persulfate, and react at 80°C for 8 hours. The product is as follows: Figure 1 shown.

[0031] Table 1 Microsphere particle size and dispersion index

[0032] Example 4 The colored microspheres of Examples 1-3 and Comparative Examples 1-2 are used to prepare a human myxovirus resistance protein A (MxA) test card. The specific steps are as follows: (1) Labeling of MxA antibodies ① Cleaning: Take 50 μL of the colored microspheres of Examples 1-3 and Comparative Examples 1-2, respectively, and add them to a centrifuge tube containing 0.5 mL of 50 mM MES (pH 6.1) buffer, mix by ultrasonication, and centrifuge to discard the supernatant; ② Activation: Add 0.5 mL of 50 mM MES (pH 6.1) buffer to the washed colored microspheres of Example 1-3 and Comparative Example 1-2, mix thoroughly by ultrasonication, add 20 μL of freshly prepared EDC (10 mg / mL) and 40 μL of NHS (10 mg / mL), vortex mix, activate at room temperature for 30 min, and centrifuge to discard the supernatant; ③ Coupling: Add 0.5 mL of 50 mM MES (pH 6.1) buffer to the activated colored microspheres of Example 1-3 and Comparative Example 1-2, and mix by ultrasonication; add 50 μg of the MxA-labeled antibody to be coupled, vortex mix, and couple at room temperature for 2 h; ④ Blocking: Add 300 μL 10% BSA and 20 μL ethanolamine to the above coupling buffer, block at room temperature for 1 hour, centrifuge and discard the supernatant; ⑤ Reconstitution: Add 500 μL of microsphere reconstitution solution to the colored microsphere-antibody complexes from Examples 1-3 and Comparative Examples 1-2 and store at 2-8°C in the dark. The microsphere reconstitution solution is formulated as follows: 10 mM PBS (pH 7.4 ± 0.05), 10 mM NaCl, 0.05% Tween-80, 1% BSA, 5% sucrose, and 0.1% ProClin 300.

[0033] (2) Preparation of nitrocellulose membrane The prepared MxA coating antibody (1 mg / mL) and goat anti-mouse IgG antibody (1 mg / mL) solutions were streaked onto nitrocellulose membranes at a coating volume of 1 μL / cm using a film streaking and gold spraying machine as the test line and quality control line, respectively, and then dried at 45°C overnight.

[0034] (3) Preparation of conjugate pad The MxA antibody complex labeled with colored microspheres of Example 1-3 and Comparative Example 1-2 in the above step (1) was sprayed onto a glass cellulose membrane at a coating volume of 5 μL / cm and dried at 45° C. overnight to prepare a conjugate pad.

[0035] (4) Reagent card assembly ① Place an absorbent pad, conjugate pad, and sample pad on the PVC substrate covered with the nitrocellulose membrane. Place the absorbent pad on one end of the control line, which rests on the nitrocellulose membrane. Place the conjugate pad on one end of the test line, which rests on the nitrocellulose membrane. Place the sample pad on top of the conjugate pad.

[0036] ② Use a chopper to cut the large plate assembled in ① into 3mm wide reagent strips, install the reagent strips into the corresponding card shells, put the installed reagent cards and a desiccant into an aluminum foil bag, seal it, and store it for later use.

[0037] (5) Reagent card test ①Tear open the seal of the aluminum foil bag and take out the test card from the aluminum foil bag.

[0038] ② Pipette 5μL of sample into the sample diluent, mix thoroughly, and then take 70μL (4 drops with a dropper) and vertically add it to the sample well of the test card. The reaction time is 8 minutes.

[0039] ③Read the test results.

[0040] Application Example 1 MxA sensitivity test Different concentrations of MxA calibrators were used for calibration curve testing. The reaction time was 8 minutes. 70 μL of diluent was added to the sample or calibrator. 80 μL of diluent was then added to the test cards prepared in Examples 1-3 and Comparative Examples 1-2. The experimental results are shown in Table 2.

[0041] Table 2 Performance comparison of microspheres in Examples 1, 2, and 3 and those in Comparative Examples 1 and 2

[0042] Note: The signal value is obtained by the card reader. The larger the value, the more obvious the color rendering.

[0043] According to the performance comparison results of the microspheres of Examples 1-3 and Comparative Examples 1-2, it can be seen that the microspheres of Examples 1-3 are significantly superior to the red microspheres of Comparative Example 1 and the blue microspheres of Comparative Example 2 in the detection performance of the MxA project. From the perspective of detection performance and visual sensory perception, the purple-red microspheres of Example 2 are preferred for the combined detection of MxA and PCT, MxA and PSP, MxA and IL-6, and MxA and HBP.

[0044] Example 6 The method for preparing the purple-red microspheres of Example 2 for MxA and PCT test cards comprises the following specific steps: (1) Labeling of MxA and PCT antibodies ① Washing: Take 50 μL of the purple-red microspheres prepared in Example 2 and add them to a centrifuge tube containing 0.5 mL of 50 mM MES (pH 6.1) buffer, mix by ultrasonication, and centrifuge to discard the supernatant; ② Activation: Add 0.5 mL of 50 mM MES (pH 6.1) buffer to the washed purple-red microspheres of Example 2, mix thoroughly by ultrasonication, add 20 μL of freshly prepared EDC (10 mg / mL) and 40 μL of NHS (10 mg / mL), vortex mix thoroughly, activate at room temperature for 30 min, and centrifuge to discard the supernatant; ③ Coupling: Add 0.5 mL of 50 mM MES (pH 6.1) buffer to the activated purple-red microspheres of Example 2 and mix thoroughly with ultrasound; add 50 μg of the MxA-labeled antibody to be coupled and 30 μg of the PCT-labeled antibody to be coupled, vortex mix thoroughly, and couple at room temperature for 2 h; ④ Blocking: Add 300 μL 10% BSA and 20 μL ethanolamine to the above coupling buffer, block at room temperature for 1 hour, centrifuge and discard the supernatant; ⑤ Reconstitution: Add 500 μL of microsphere reconstitution solution to the purple microsphere-antibody complex from Example 2 and store at 2-8°C in the dark. The microsphere reconstitution solution formula is: 10 mM PBS (pH 7.4 ± 0.05), 10 mM NaCl, 0.05% Tween-80, 1% BSA, 5% sucrose, and 0.1% ProClin 300.

[0045] (2) Preparation of nitrocellulose membrane The prepared MxA coated antibody, PCT coated antibody (1 mg / mL) and goat anti-mouse IgG antibody (1 mg / mL) solution was drawn on the nitrocellulose membrane with a coating amount of 1 μL / cm by using a membrane scribing and gold spraying integrated machine as a detection line and a quality control line, respectively, and was dried at 45°C overnight.

[0046] (3) Preparation of a conjugate pad The purple microsphere-labeled MxA antibody, PCT antibody complex in step (1) above was sprayed on a glass fiber membrane with a coating amount of 5 μL / cm, and was dried at 45°C overnight to prepare a conjugate pad.

[0047] (4) Assembly of a reagent card ①The above coated nitrocellulose membrane was pasted on a PVC bottom plate in turn with a water absorption pad, a conjugate pad and a sample pad. The quality control line was pasted with a water absorption pad at one end, the water absorption pad was placed on the NC membrane, the detection line was pasted with a conjugate pad at one end, the conjugate pad was placed on the NC membrane at one end, and the sample pad was placed on the conjugate pad.

[0048] ②The large plate assembled in ① was cut into a reagent strip with a width of 3 mm by using a cutting machine, the reagent strip was loaded into a card shell, the assembled reagent card and a desiccant were placed in an aluminum foil bag for sealing, and were stored for standby use.

[0049] (5) Detection of a reagent card ①The aluminum foil bag was opened, and the detection card was taken out from the aluminum foil bag.

[0050] ②5 μL of the sample was taken and added to the sample diluent, 70 μL (4 drops of a dropper) of the mixture was added vertically to the sample addition hole of the detection card after being fully mixed, and the reaction time was 8 min.

[0051] ③The detection result was read.

[0052] The product of the present embodiment and the same product approved for marketing were subjected to comparative study on 212 clinical whole blood samples, the MxA positive coincidence rate was 96.15%, the negative coincidence rate was 93.28%, and the total coincidence rate was 94.34%; the PCT positive coincidence rate was 95.52%, the negative coincidence rate was 91.72%, and the total coincidence rate was 92.92%, and the test result showed that the coincidence rate was good.

[0053] Example 7 The purple microspheres of Example 2 were used for the preparation method of MxA and PSP detection cards, and the specific steps were as follows: (1) Labeling of MxA and PSP antibodies ①Washing: 50 μL of the purple microspheres of Example 2 were taken and added to a centrifugal tube containing 0.5 mL of 50 mM MES (pH 6.1) buffer, and were ultrasonically mixed and centrifuged to discard the supernatant; ② Activation: Add 0.5 mL of 50 mM MES (pH 6.1) buffer to the washed purple-red microspheres of Example 2, mix thoroughly by ultrasonication, add 20 μL of freshly prepared EDC (10 mg / mL) and 40 μL of NHS (10 mg / mL), vortex mix thoroughly, activate at room temperature for 30 min, and centrifuge to discard the supernatant; ③ Coupling: Add 0.5 mL of 50 mM MES (pH 6.1) buffer to the activated purple-red microspheres of Example 2 and mix thoroughly with ultrasound; add 50 μg of the MxA-labeled antibody to be coupled and 50 μg of the PSP-labeled antibody to be coupled, vortex mix thoroughly, and couple at room temperature for 2 h; ④ Blocking: Add 300 μL 10% BSA and 20 μL ethanolamine to the above coupling buffer, block at room temperature for 1 hour, centrifuge and discard the supernatant; ⑤ Reconstitution: Add 500 μL of microsphere reconstitution solution to the purple microsphere-antibody complex from Example 2 and store at 2-8°C in the dark. The microsphere reconstitution solution formula is: 10 mM PBS (pH 7.4 ± 0.05), 10 mM NaCl, 0.05% Tween-80, 1% BSA, 5% sucrose, and 0.1% ProClin 300.

[0054] (2) Preparation of nitrocellulose membrane The prepared MxA coating antibody (1 mg / mL), PSP coating antibody (2 mg / mL), and goat anti-mouse IgG antibody (1 mg / mL) solutions were streaked onto nitrocellulose membranes at a coating volume of 1 μL / cm using a film streaking and gold spraying machine as the test line and quality control line, respectively, and dried at 45°C overnight.

[0055] (3) Preparation of conjugate pad The purple-red microsphere-labeled MxA antibody and PSP antibody complex in step (1) above was sprayed onto the glass cellulose membrane at a coating volume of 5 μL / cm and dried at 45°C overnight to prepare a conjugate pad.

[0056] (4) Reagent card assembly ① Place an absorbent pad, conjugate pad, and sample pad on the PVC substrate covered with the nitrocellulose membrane. Place the absorbent pad on one end of the control line, which rests on the nitrocellulose membrane. Place the conjugate pad on one end of the test line, which rests on the nitrocellulose membrane. Place the sample pad on top of the conjugate pad.

[0057] ② Use a chopper to cut the large plate assembled in ① into 3mm wide reagent strips, install the reagent strips into the corresponding card shells, put the installed reagent cards and a desiccant into an aluminum foil bag, seal it, and store it for later use.

[0058] (5) Reagent card test ①Tear open the seal of the aluminum foil bag and take out the test card from the aluminum foil bag.

[0059] ② Pipette 5μL of sample into the sample diluent, mix thoroughly, and then take 70μL (4 drops with a dropper) and vertically add it to the sample well of the test card. The reaction time is 8 minutes.

[0060] ③Read the test results.

[0061] The product of this embodiment was compared with similar products approved for marketing in 202 clinical whole blood samples. The MxA positive compliance rate was 96.92%, the negative compliance rate was 92.7%, and the overall compliance rate was 94.06%. The PSP positive compliance rate was 93.52%, the negative compliance rate was 91.42%, and the overall compliance rate was 92.92%. The test results showed a good compliance rate.

[0062] Example 8 The method for preparing the purple-red microspheres of Example 2 for the MxA and IL-6 detection card comprises the following specific steps: (1) Labeling of MxA and IL-6 antibodies ① Washing: Take 50 μL of the purple-red microspheres prepared in Example 2 and add them to a centrifuge tube containing 0.5 mL of 50 mM MES (pH 6.1) buffer, mix by ultrasonication, and centrifuge to discard the supernatant; ② Activation: Add 0.5 mL of 50 mM MES (pH 6.1) buffer to the washed purple-red microspheres of Example 2, mix thoroughly by ultrasonication, add 20 μL of freshly prepared EDC (10 mg / mL) and 40 μL of NHS (10 mg / mL), vortex mix thoroughly, activate at room temperature for 30 min, and centrifuge to discard the supernatant; ③ Coupling: Add 0.5 mL of 50 mM MES (pH 6.1) buffer to the activated purple-red microspheres of Example 2 and mix thoroughly with ultrasound; add 50 μg of the MxA-labeled antibody to be coupled and 50 μg of the IL-6-labeled antibody to be coupled, vortex mix thoroughly, and couple at room temperature for 2 h; ④ Blocking: Add 300 μL 10% BSA and 20 μL ethanolamine to the above coupling buffer, block at room temperature for 1 hour, centrifuge and discard the supernatant; ⑤ Reconstitution: Add 500 μL of microsphere reconstitution solution to the purple microsphere-antibody complex from Example 2 and store at 2-8°C in the dark. The microsphere reconstitution solution formula is: 10 mM PBS (pH 7.4 ± 0.05), 10 mM NaCl, 0.05% Tween-80, 1% BSA, 5% sucrose, and 0.1% ProClin 300.

[0063] (2) Preparation of nitrocellulose membrane The prepared MxA coating antibody (1 mg / mL), IL-6 coating antibody (1.5 mg / mL), and goat anti-mouse IgG antibody (1 mg / mL) solutions were streaked onto nitrocellulose membranes at a coating volume of 1 μL / cm using a film streaking and gold spraying machine as the test line and quality control line, respectively, and dried at 45°C overnight.

[0064] (3) Preparation of conjugate pad The purple-red microsphere-labeled MxA antibody and IL-6 antibody complex in step (1) above was sprayed onto the glass cellulose membrane at a coating volume of 5 μL / cm and dried at 45°C overnight to prepare a conjugate pad.

[0065] (4) Reagent card assembly ① Place an absorbent pad, conjugate pad, and sample pad on the PVC substrate covered with the nitrocellulose membrane. Place the absorbent pad on one end of the control line, which rests on the nitrocellulose membrane. Place the conjugate pad on one end of the test line, which rests on the nitrocellulose membrane. Place the sample pad on top of the conjugate pad.

[0066] ② Use a chopper to cut the large plate assembled in ① into 3mm wide reagent strips, install the reagent strips into the corresponding card shells, put the installed reagent cards and a desiccant into an aluminum foil bag, seal it, and store it for later use.

[0067] (5) Reagent card test ①Tear open the seal of the aluminum foil bag and take out the test card from the aluminum foil bag.

[0068] ② Pipette 5μL of sample into the sample diluent, mix thoroughly, and then take 70μL (4 drops with a dropper) and vertically add it to the sample well of the test card. The reaction time is 8 minutes.

[0069] ③Read the test results.

[0070] The product of this embodiment was compared with similar products approved for marketing in 225 clinical whole blood samples. The MxA positive compliance rate was 94.2%, the negative compliance rate was 92.31%, and the overall compliance rate was 92.89%. The IL-6 positive compliance rate was 94.37%, the negative compliance rate was 92.21%, and the overall compliance rate was 92.89%. The test results showed a good compliance rate.

[0071] Example 9 The method for preparing the purple-red microspheres of Example 2 for use in MxA and HBP detection cards comprises the following specific steps: (1) Labeling of MxA and HBP antibodies ① Washing: Take 50 μL of the purple-red microspheres prepared in Example 2 and add them to a centrifuge tube containing 0.5 mL of 50 mM MES (pH 6.1) buffer, mix by ultrasonication, and centrifuge to discard the supernatant; ② Activation: Add 0.5 mL of 50 mM MES (pH 6.1) buffer to the washed purple-red microspheres of Example 2, mix thoroughly by ultrasonication, add 20 μL of freshly prepared EDC (10 mg / mL) and 40 μL of NHS (10 mg / mL), vortex mix thoroughly, activate at room temperature for 30 min, and centrifuge to discard the supernatant; ③ Coupling: Add 0.5 mL of 50 mM MES (pH 6.1) buffer to the activated purple-red microspheres of Example 2 and mix thoroughly with ultrasound; add 50 μg of the MxA-labeled antibody to be coupled and 30 μg of the HBP-labeled antibody to be coupled, vortex mix thoroughly, and couple at room temperature for 2 h; ④ Blocking: Add 300 μL 10% BSA and 20 μL ethanolamine to the above coupling buffer, block at room temperature for 1 hour, centrifuge and discard the supernatant; ⑤ Reconstitution: Add 500 μL of microsphere reconstitution solution to the purple microsphere-antibody complex from Example 2 and store at 2-8°C in the dark. The microsphere reconstitution solution formula is: 10 mM PBS (pH 7.4 ± 0.05), 10 mM NaCl, 0.05% Tween-80, 1% BSA, 5% sucrose, and 0.1% ProClin 300.

[0072] (2) Preparation of nitrocellulose membrane The prepared MxA coating antibody (1 mg / mL), HBP coating antibody (1 mg / mL), and goat anti-mouse IgG antibody (1 mg / mL) solutions were streaked onto nitrocellulose membranes at a coating volume of 1 μL / cm using a film streaking and gold spraying machine as the test line and quality control line, respectively, and then dried at 45°C overnight.

[0073] (3) Preparation of conjugate pad The purple-red microsphere-labeled MxA antibody and HBP antibody complex in step (1) above was sprayed onto the glass cellulose membrane at a coating volume of 5 μL / cm and dried at 45°C overnight to prepare a conjugate pad.

[0074] (4) Reagent card assembly ① Place an absorbent pad, conjugate pad, and sample pad on the PVC substrate covered with the nitrocellulose membrane. Place the absorbent pad on one end of the control line, which rests on the nitrocellulose membrane. Place the conjugate pad on one end of the test line, which rests on the nitrocellulose membrane. Place the sample pad on top of the conjugate pad.

[0075] ② Use a chopper to cut the large plate assembled in ① into 3mm wide reagent strips, install the reagent strips into the corresponding card shells, put the installed reagent cards and a desiccant into an aluminum foil bag, seal it, and store it for later use.

[0076] (5) Reagent card test ①Tear open the seal of the aluminum foil bag and take out the test card from the aluminum foil bag.

[0077] ② Pipette 5μL of sample into the sample diluent, mix thoroughly, and then take 70μL (4 drops with a dropper) and vertically add it to the sample well of the test card. The reaction time is 8 minutes.

[0078] ③Read the test results.

[0079] The product of this embodiment was compared with similar products approved for marketing in 214 clinical whole blood samples. The MxA positive compliance rate was 95.45%, the negative compliance rate was 92.57%, and the overall compliance rate was 93.46%. The HBP positive compliance rate was 93.94%, the negative compliance rate was 91.22%, and the overall compliance rate was 92.06%. The test results showed a good compliance rate.

[0080] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing dual-color synergistic colored microspheres, characterized in that: The following steps are involved: (1) Preparation of red silica microsphere cores: pure water and anhydrous ethanol were added to a reaction vessel, and red oil-soluble dye was added. After ultrasonic dissolution, the reaction vessel was fixed on an electric stirrer for stirring, and ammonia water was added. After a certain period of time, the silane coupling agent TEOS was added. The reaction was allowed to react at room temperature overnight. After the reaction was completed, the mother liquor was removed by centrifugation, and pure water was added for ultrasonic dispersion and then set aside. (2) Coating with a blue polymer shell: add pure water to the reaction vessel, pass nitrogen to remove oxygen, add anionic surfactant and stir to dissolve, add the red silica microsphere core prepared in step (1); weigh the blue oil-soluble dye and dissolve it in the mixed solution of purified styrene and functional monomer, slowly add it to the reaction vessel, stir at high speed to emulsify, heat to 60°C, add potassium persulfate, and react at 75-85°C for 4-12 hours.

2. The method for preparing dual-color synergistic colored microspheres according to claim 1, characterized in that: In the step (1), the volume ratio of pure water to anhydrous ethanol is 1:4-9.

3. The method for preparing dual-color synergistic colored microspheres according to claim 1, characterized in that: In the step (1), the red oil-soluble dye includes solvent red or disperse red.

4. The method for preparing dual-color synergistic colored microspheres according to claim 1, characterized in that: In the step (1), the mass ratio of the red oil-soluble dye to the silane coupling agent TEOS is 1:2-3.

5. The method for preparing dual-color synergistic colored microspheres according to claim 1, characterized in that: In the step (2), the blue oil-soluble dye includes disperse blue and solvent blue.

6. The method for preparing dual-color synergistic colored microspheres according to claim 1, characterized in that: In the step (2), the mass ratio of the blue oil-soluble dye to all monomers is 1:10-50; the anionic surfactant is sodium lauryl sulfate; the functional monomer is methacrylic acid or acrylic acid, wherein the volume ratio of styrene to the functional monomer is 1:0.04-0.

2.

7. The method for preparing dual-color synergistic colored microspheres according to claim 6, characterized in that: The functional monomer is methacrylic acid.

8. Double-color synergistic colored microspheres prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the dual-color synergistic colored microspheres according to claim 8 in high-sensitivity single-link detection or multiple-link detection of infection markers.

10. Use of the dual-color synergistic colored microspheres according to claim 9 in high-sensitivity single-link detection or multiple-link detection of infection markers, characterized in that: The infection markers are viral infection markers and bacterial infection markers. The viral infection marker is MxA protein, and the bacterial infection markers include CRP, PCT, PSP, IL-6 and HBP.

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